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The HIV-1 virion-associated protein vpr is a coactivator of the human glucocorticoid receptor.

Kino T, Gragerov A, Kopp JB, Stauber RH, Pavlakis GN, Chrousos GP - J. Exp. Med. (1999)

Bottom Line: We report that Vpr enhances the activity of glucocorticoids in lymphoid and muscle-derived cell lines by interacting directly with the glucocorticoid receptor and general transcription factors, acting as a coactivator.A mutant Vpr molecule with disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsive genes and became a dominant-negative inhibitor of Vpr, possibly by retaining its general transcription factor-binding activities.The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS.

View Article: PubMed Central - PubMed

Affiliation: Section on Pediatric Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA. kinot@cc1.nichd.nih.gov

ABSTRACT
The HIV-1 virion-associated accessory protein Vpr affects both viral replication and cellular transcription, proliferation, and differentiation. We report that Vpr enhances the activity of glucocorticoids in lymphoid and muscle-derived cell lines by interacting directly with the glucocorticoid receptor and general transcription factors, acting as a coactivator. Vpr contains the signature motif LXXLL also present in cellular nuclear receptor coactivators, such as steroid receptor coactivator 1 and p300/CREB-binding protein, which mediates their interaction with the glucocorticoid and other nuclear hormone receptors. A mutant Vpr molecule with disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsive genes and became a dominant-negative inhibitor of Vpr, possibly by retaining its general transcription factor-binding activities. The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS.

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Vpr enhances the GR coactivation by p300 or SRC-1.  A204 cells were transfected with 1.5 μg/well of pMMTV-luc, 0.6 μg/ well of pCDNA3-VPR, 2.0 μg/well of CMVβ-p300-CHA, or 2.0 μg/ well of pCR-SRC-1a as indicated. pCDNA3 (instead of pCDNA3-VPR  and pCMVβ-p300-CHA) or pCR3.1 (instead of pCR-SRC-1a) was  cotransfected as needed to maintain the same amount of DNA. Black and  white bars, mean ± SEM values obtained in the absence or presence of  10−7 M dexamethasone, respectively.
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Figure 6: Vpr enhances the GR coactivation by p300 or SRC-1. A204 cells were transfected with 1.5 μg/well of pMMTV-luc, 0.6 μg/ well of pCDNA3-VPR, 2.0 μg/well of CMVβ-p300-CHA, or 2.0 μg/ well of pCR-SRC-1a as indicated. pCDNA3 (instead of pCDNA3-VPR and pCMVβ-p300-CHA) or pCR3.1 (instead of pCR-SRC-1a) was cotransfected as needed to maintain the same amount of DNA. Black and white bars, mean ± SEM values obtained in the absence or presence of 10−7 M dexamethasone, respectively.

Mentions: We also studied the interactions of Vpr with GR and components of the transcription complex in dexamethasone- and mock-treated pCMV-FLAG-VPR–transfected cells by coimmunoprecipitations of cell extracts. As shown in Fig. 5, FLAG-Vpr was coimmunoprecipitated by anti-TFIID (TBP), anti-TFIIB, or anti-GR antibodies in dexamethasone-treated cells, suggesting that Vpr binds to components of the transcription machinery and to the GR, as part of the glucocorticoid-activated transcription initiation complex (23). FLAG-VprL64A was coimmunoprecipitated by anti-TFIIB antibody, suggesting that the TFIIB-binding site of this mutant remains functional, whereas FLAG-Vpr(36–96) was not precipitated by either GR, TFIID, or TFIIB antibodies. Coprecipitation of FLAG-Vpr was not efficient by TFIIB antibodies in the absence of dexamethasone, whereas it increased in its presence. FLAG-VprL64A was similarly precipitated by TFIIB antibodies, but this did not increase in the presence of dexamethasone. These results suggest that Vpr binds directly to TFIIB through the NH2-terminal part of the molecule and to the GR through the LXXLL coactivator domain. Binding to both factors leads to enhanced incorporation of Vpr into a large transcription complex also including other transcription factors such as TFIID. The binding of the VprL64A mutant to TFIIB but not to the GR may explain its transdominant negative phenotype as competition of the mutant with wild-type Vpr for the TFIIB-binding site. If Vpr becomes part of a bigger transcription complex in the presence of dexamethasone, then it may be coprecipitated with antibodies for other proteins known to be in the GR transcription complex, such as the coregulators p300. In coimmunoprecipitation experiments using lysate of FLAG-Vpr–transfected A204 cells, Vpr and p300 were either weakly or strongly coprecipitated by anti-FLAG antibody in the absence or presence of dexamethasone, respectively (data not shown). This may reflect the presence of Vpr and p300 in the same complex. Alternatively, it may indicate additional contacts of Vpr with p300. It was recently suggested that Vpr transactivation on the HIV promoter is mediated through p300 (45). To study any potential interactions of Vpr and p300, we compared the effects of transfected Vpr and p300/CBP coregulators on the MMTV promoter (Fig. 6 A). We also studied the interaction of Vpr with another GR coactivator, steroid receptor coactivator (SRC)-1, by cotransfecting pCDNA3-VPR with an SRC-1 expression vector. As shown in Fig. 6, A and B, Vpr potentiated dexamethasone activity >20-fold. Vpr and p300 or SRC-1a synergistically enhanced ligand-activated GR activity on the MMTV promoter. No enhancement was observed in the absence of glucocorticoid. Therefore, Vpr appears to synergize with other coactivators in the activation of the MMTV promoter.


The HIV-1 virion-associated protein vpr is a coactivator of the human glucocorticoid receptor.

Kino T, Gragerov A, Kopp JB, Stauber RH, Pavlakis GN, Chrousos GP - J. Exp. Med. (1999)

Vpr enhances the GR coactivation by p300 or SRC-1.  A204 cells were transfected with 1.5 μg/well of pMMTV-luc, 0.6 μg/ well of pCDNA3-VPR, 2.0 μg/well of CMVβ-p300-CHA, or 2.0 μg/ well of pCR-SRC-1a as indicated. pCDNA3 (instead of pCDNA3-VPR  and pCMVβ-p300-CHA) or pCR3.1 (instead of pCR-SRC-1a) was  cotransfected as needed to maintain the same amount of DNA. Black and  white bars, mean ± SEM values obtained in the absence or presence of  10−7 M dexamethasone, respectively.
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Related In: Results  -  Collection

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Figure 6: Vpr enhances the GR coactivation by p300 or SRC-1. A204 cells were transfected with 1.5 μg/well of pMMTV-luc, 0.6 μg/ well of pCDNA3-VPR, 2.0 μg/well of CMVβ-p300-CHA, or 2.0 μg/ well of pCR-SRC-1a as indicated. pCDNA3 (instead of pCDNA3-VPR and pCMVβ-p300-CHA) or pCR3.1 (instead of pCR-SRC-1a) was cotransfected as needed to maintain the same amount of DNA. Black and white bars, mean ± SEM values obtained in the absence or presence of 10−7 M dexamethasone, respectively.
Mentions: We also studied the interactions of Vpr with GR and components of the transcription complex in dexamethasone- and mock-treated pCMV-FLAG-VPR–transfected cells by coimmunoprecipitations of cell extracts. As shown in Fig. 5, FLAG-Vpr was coimmunoprecipitated by anti-TFIID (TBP), anti-TFIIB, or anti-GR antibodies in dexamethasone-treated cells, suggesting that Vpr binds to components of the transcription machinery and to the GR, as part of the glucocorticoid-activated transcription initiation complex (23). FLAG-VprL64A was coimmunoprecipitated by anti-TFIIB antibody, suggesting that the TFIIB-binding site of this mutant remains functional, whereas FLAG-Vpr(36–96) was not precipitated by either GR, TFIID, or TFIIB antibodies. Coprecipitation of FLAG-Vpr was not efficient by TFIIB antibodies in the absence of dexamethasone, whereas it increased in its presence. FLAG-VprL64A was similarly precipitated by TFIIB antibodies, but this did not increase in the presence of dexamethasone. These results suggest that Vpr binds directly to TFIIB through the NH2-terminal part of the molecule and to the GR through the LXXLL coactivator domain. Binding to both factors leads to enhanced incorporation of Vpr into a large transcription complex also including other transcription factors such as TFIID. The binding of the VprL64A mutant to TFIIB but not to the GR may explain its transdominant negative phenotype as competition of the mutant with wild-type Vpr for the TFIIB-binding site. If Vpr becomes part of a bigger transcription complex in the presence of dexamethasone, then it may be coprecipitated with antibodies for other proteins known to be in the GR transcription complex, such as the coregulators p300. In coimmunoprecipitation experiments using lysate of FLAG-Vpr–transfected A204 cells, Vpr and p300 were either weakly or strongly coprecipitated by anti-FLAG antibody in the absence or presence of dexamethasone, respectively (data not shown). This may reflect the presence of Vpr and p300 in the same complex. Alternatively, it may indicate additional contacts of Vpr with p300. It was recently suggested that Vpr transactivation on the HIV promoter is mediated through p300 (45). To study any potential interactions of Vpr and p300, we compared the effects of transfected Vpr and p300/CBP coregulators on the MMTV promoter (Fig. 6 A). We also studied the interaction of Vpr with another GR coactivator, steroid receptor coactivator (SRC)-1, by cotransfecting pCDNA3-VPR with an SRC-1 expression vector. As shown in Fig. 6, A and B, Vpr potentiated dexamethasone activity >20-fold. Vpr and p300 or SRC-1a synergistically enhanced ligand-activated GR activity on the MMTV promoter. No enhancement was observed in the absence of glucocorticoid. Therefore, Vpr appears to synergize with other coactivators in the activation of the MMTV promoter.

Bottom Line: We report that Vpr enhances the activity of glucocorticoids in lymphoid and muscle-derived cell lines by interacting directly with the glucocorticoid receptor and general transcription factors, acting as a coactivator.A mutant Vpr molecule with disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsive genes and became a dominant-negative inhibitor of Vpr, possibly by retaining its general transcription factor-binding activities.The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS.

View Article: PubMed Central - PubMed

Affiliation: Section on Pediatric Endocrinology, Developmental Endocrinology Branch, National Institute of Child Health and Human Development, Bethesda, MD 20892, USA. kinot@cc1.nichd.nih.gov

ABSTRACT
The HIV-1 virion-associated accessory protein Vpr affects both viral replication and cellular transcription, proliferation, and differentiation. We report that Vpr enhances the activity of glucocorticoids in lymphoid and muscle-derived cell lines by interacting directly with the glucocorticoid receptor and general transcription factors, acting as a coactivator. Vpr contains the signature motif LXXLL also present in cellular nuclear receptor coactivators, such as steroid receptor coactivator 1 and p300/CREB-binding protein, which mediates their interaction with the glucocorticoid and other nuclear hormone receptors. A mutant Vpr molecule with disruption of this coactivator signature motif lost its ability to influence transcription of glucocorticoid-responsive genes and became a dominant-negative inhibitor of Vpr, possibly by retaining its general transcription factor-binding activities. The glucocorticoid coactivator activity of Vpr may contribute to increased tissue glucocorticoid sensitivity in the absence of hypercortisolism and to the pathogenesis of AIDS.

Show MeSH
Related in: MedlinePlus